Wiring diagram. A.-F. Miller, 2008, pg 1. Garrett & Grisham Fig

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1 Wiring diagram 1 Garrett & Grisham Fig

2 Gradients as signalling devices Nerve impulses propagated at 100 m/s Neuronal impulses are carried along axons as electrical signals: transient changes in the axon membrane polarization (membrane potential). In the neuron: 50 mm Na +, 400 mm K +, 60 mm l - Surrounding: 400 mm Na +, 20 mm K +, 560 mm l - Inside potential is -60 mv relative to outside. Upon transient local influx of a cation, the potential decreases in size to -40 mv (depolarization). Local voltage-gated channels open, Na + enters (and the potential rises). 2 DMIN effect Garrett & Grisham Fig

3 Propagation of an action potential When the membrane potential goes +ve (+30 mv), K + channels open, K + rushes out, potential drops again and the ATPases get to work restoring the ion gradients. The action potential moves fast without attenuation. 3 Garrett & Grisham Fig

4 Transmission of action potential At synaptic terminus of axon, action potential triggers opening of voltage-gated a 2+ channels. Influx of a 2+ triggers exocytosis of vesicles filled with acetyl choline. Acetyl choline diffuses across the synapse and initiates an impulse in the next neuron. 4

5 Zn 2+, a spectroscopicallychallenged transition metal ion Similarly to Mg 2+, Zn 2+ serves both structural and catalytic roles. d 10 electronic configuration of Zn 2+ means that it is not dependent on specific coordination geometry, the geometry can instead accommodate the charge and size of the ligands. Tetrahedral coordination preferred. Tetrahedral (Td) Zn is a stronger Lewis acid than octahedral (h) would be. Zn 2+ is intermediate in hardness: ligands are, N and S. 5

6 Zn 2+ enzymes Peptidases, amidases, peptidases, hydrolases, lactamase, phosphodiesterases. Table 12.1 Enzyme arbonic Anhydrase His X His 22X His β lactamase His X His 121X His thermolysin His 3X His 19X Glu carboxypeptidase His 2X Glu 123X His alcohol dehydrogenase ys 20X His 106X ys alkaline phosphatase Asp 3X His 80X His adenosine deaminase His X His 196X His 6

7 Activation of coordinated water The pk of coordinated water drops to 7, and the resulting H - is a strong nucleophile. Ionization of coordinated H2 to give H -, polarization of coordinated H2, H2 as a labile place-holder. ALS: the possibility of binding several groups at once permits Zn 2+ to bring together reactants (proximity effect). Polarization of other ligands/reactants. 7

8 arbonic Anhydrase Turns over at 10 9 s -1, 2 + H2 H + + H3 - - Zn 2+ N-His H H + :B - Zn 2+ N-His H H 2 H 3 - Zn 2+ H 2 N-His Proximity, electrostatic, nucleophillic, general base (His64) 8

9 Peptidases Thermolysins (an endopeptidase) and carboxypeptidases are early examples. They are also the reason we include EDTA in our cell lysis buffers. Gen. Base Glu- - H + H Arg + Gen. Acid NH Glu-!"!+ HR'!+ H - - Zn 2+ H R -Glu - + Arg ~ Arg + - H H R NH - Zn 2+!" -Glu - HR' + Arg Tetrahedral intermediate ~ Glu- - Arg + - H Zn 2+ H R NH H -Glu - H 2 HR' ~ + Arg Nucleophilic attack of water on promoted by Zn 2+, assisted by Glu -. 9 Td intermediate stabilized by Arg, Glu, Zn 2+. Product displacement by H2.

10 Liver Alcohol Dehydrogenase 10 Figure 12.6

11 Ada protein: a super-ys. 11

12 metallo-β-lactamases: agents of drug-resistance R R R NH H H HN S H H 3 H 3 - NH H Ser(enzyme) H NH S H H 3 H 3 - NH H H NH H S H H 3 H 3 - Glycoprotein transpeptidase involved in cell wall biosynthesis.! lactamase activates water as the attacking group. 12

13 Multinuclear Zn sites: alkaline Phosphatase. 13

14 Zn fingers 14 1p47.pdb E.PEISAH,..PAB (2003) J.ML.BIL. 330:1

15 Zn fingers 15 1p47.pdb E.PEISAH,..PAB (2003) J.ML.BIL. 330:1

16 Zn fingers 16 1p47.pdb E.PEISAH,..PAB (2003) J.ML.BIL. 330:1

17 Zn fingers 17 1p47.pdb E.PEISAH,..PAB (2003) J.ML.BIL. 330:1

18 Zn fingers 18 1p47.pdb E.PEISAH,..PAB (2003) J.ML.BIL. 330:1

19 19 Sample problems Metal ion and oxidation state d-electron count. High spin vs. low spin electronic configuration in h and Td coordination. Identities of coordinating amino acids, and modes of binding. Hard-soft series and ligand-metal preferred pairing. General schemes for metal acquisition. Non-redox cations Na +, K +, a 2+, Mg 2+. Forms in which they occur, roles they play, issues they raise. Why? Zn 2+, the simple transition metal ion: what does it do? Why?

20 Example Uranyl ion (U2 2+ ) is commonly used to form heavy-atom derivatives for X-ray diffraction studies. Given that this is a relatively hard ion, what sorts of functional groups would you expect to be derivatized? How does this behaviour compare with that expected for another heavy-metal derivatizing ion, Hg 2+? 20

21 More examples Draw a Glu side chain with a metal ion coordinated to it. What metal ions are likely to bind to His? Ammonia and the tertiary amines in EDTA are common ligands to metal ions, yet the terminal amine of Lys is only rarely a ligand to metal ions bound to proteins in aqueous solution. Propose an explanation for this. 21